Thyroid hormones (THs) play key regulatory roles in growth, development and metabolism in vertebrates. Modulation of the cellular hormonal response is largely through the activity of two nuclear TH receptors, TRα and TRβ, which act as transcription factors and alter gene expression programs. Little information is available regarding their structure and regulation in reptiles. We have cloned the expressed sequences encoding these two receptors in the American alligator, Alligator mississippiensis. The encoded putative proteins share a high degree of amino acid sequence conservation with other vertebrates, however, both alligator TRs contain putative N-terminal truncations. This phenomenon is shared with the chicken for TRβ, but not for TRα, making this the first demonstration of this type of TRα isoform. We measured the steady-state levels of TR transcripts in heart, lung, liver, thyroid, cliterophallus/phallus, and gonad of juvenile alligators 24 and 48 h after injection with thyroid stimulating hormone (TSH). TRα transcript levels were increased in the heart, decreased in the lung and cliterophallus/phallus, and unaffected in the liver, thyroid, and gonad. TRβ transcript levels were increased in the heart, lung, and gonad whereas estrogen receptor α transcript levels were elevated by TSH treatment only in the gonad. Modulation of these transcripts in the gonad is consistent with TH playing an important role in this tissue’s function since seasonal TH fluctuations coincide with reproductive events. These data demonstrate that alligator tissues are differentially responsive to TSH by regulation of TR expression and provide an important comparative framework among vertebrates.